Process for recovering flavonoids from bark



United States Patent Shelton, Wash, assignor to Rayonier Shelton, Wash,a corporation of Dela- Herbert L. Hergert,

Incorporated, ware No Drawing. Application June 20, 1956 Serial No.592,495

8 Claims. (Cl. 260-3452) This inventionrelates to a process forrecovering the fiavonoids contained in bark and, more particularly, tothe recovery and purification of crystalline myricetin from theflavonoid extractive of lodgepole pine bark. The invention isparticularly suited to the separate recovery of myricetin from a mixturecontaining myricetin and other fiavonoids.

The bark of certain conifers, such as Jeffrey pine (Pinus jefireyi), andDouglas fir (Pseudotsuga elliotti) has been found to containconsiderable quantities of fiavonoids, the principal constituent ofwhich is dihydroquercetin. It has now been discovered that the fiavonol,myricetin (3,3,4,5,5',7-hexahydroxyfiavone), can be recovered insubstantial quantities from the bark of lodgepole pine (Pinus contortaDougl.).

In an extensive investigation of the composition of the flavonoid'fraction isolated from the bark of lodgepole pine, I have discoveredthat myricetin comprises approximately 2 percent by weight of the bark,and accounts for about 90 percent of the total fiavonoids in the bark.The myricetin present in lodgepole pine bark seems to exist in the formof the uncombined fiavonol, and may be extracted with suitable solventsalong with other fiavonoids in a flavonoid fraction of the bark. Upondissolving this fiavonoid fraction in a solvent, I have found thatmyricetin can be selectively crystallized in the presence of theremaining dissolved fiavonoids. Alternatively, the flavonoid fractioncan be triturated with a solvent so that the insoluble crystallineresidue remaining undissolved consists of nearly pure myricetin, whilethe balance of the fiavonoids, comprising quercetin, ampelopsin(dihydromyricetin), dihydroquercetin and traces of flavonoid impurities,are selectively dissolved and removed by the triturating solvent.

Based on these discoveries, the invention provides a process forrecovering crystalline myricetin from lodgepole pine bark whichcomprises extracting a mixture of fiavonoids from sub-divided lodgepolepine bark, dissolving the mixture of fiavonoids in a solvent, and thenselectively crystallizing myricetin from the solvent in the presence ofthe remaining dissolved fiavonoids. As an advantageous alternative, themyricetin may be recovered by trituration of the flavonoid fraction witha solvent, thereby dissolving the fiavonoid impurities, such asquercetin, ampelopsin and dihydroqu'ercetin, and leaving substantiallypure myricetin as a crystalline residue.

Upon removal of crystalline myricetin from the solvent, quercetin may beselectively precipitated. Thus, when the triturating solvent is acetone,dilution of the triturating solvent with water, followed by cooling,causes quercetin to selectively precipitate from the resultant solutionwhile the water-soluble flavanones remain dissolved. Following thecrystallization of quercetin, the dissolved flavanones may beprecipitated by the addition of a fiavanone precipitating agent, such asdibasic potassium phosphate, and recovered by acidification of thepotassium salts of the flavanones. In this 2,870,165 Patented Jan. 20,1959 manner, substantially all of the fiavones and fiavanones may beseparately recovered from the fiavonoid fraction of lodgepole pine bark.

Lodgepol'e pine is a tree indigenous to the Pacific Northwest and mostof the Rocky Mountain region of the United States. Although someauthorities distinguish two varieties .of this pine, one of which islimited to the coastal region (Pinus contorta var. Contorta), and theother to the interior (Pinus contorta var. latifolia Engelm.), there isno significantdifference in the chemical nature of the extractivesisolated fromthe bark of the several varieties. There is, however, aslight difference in the quantity of fiavonoids extracted, for the barkof the coastal variety of lodgepole pine contains appreciably largeramounts of fiavonoids. The following table illustrates the range inquantity of total fiavonoids contained in the bark of lodgepole pinegrowing in several geographical areas:

Grays Harbor, Washington 2. Mt. Hood National Forest, Oreg 0. OchochoNational Forest, Oreg 1.

In a preferred practical embodiment of the invention, the lodgepole pinebark is air dried and reduced to a suitable state of sub-division, as ina hammer mill, to facilitate extraction. Although solvent extraction maybe performed on various sizes of bark particles, the efficiency of theextraction is significantly increased if the bark has been ground to astate that will permit rapid penetration of the solvents.

To obtain the flavonoid fraction, the bark is preferably first extractedwith a suitable solvent capable of dissolving not only the fiavonoidsbut waxes and fatty material as well. Among such solvents arelow-molecular weight ethers, such as ethyl ether, low-molecular weightketones, such as acetone and methyl ethyl ketone, and lowmolecularweight esters, such as ethyl acetate. The solvent extract is dried byevaporation of the solvent, leaving a mixture containing not only theflavonoid fraction of the bark, but the fatty material and waxes. Upontreatment of this extract with a defatting solvent, capable ofdissolving the waxes and fatty material but not the fiavonoids, only aninsoluble flavonoid residue remains. By paper chromatographicfractionation of the fiavonoid residue, dissolved in an aceticacid-water (60:40) solvent system, the following fiavonoids wereidentified:

The separation of fatty material and waxesfrom the bark extract,containing both fatty material and flavonoids, may be accomplished by avariety of defatting solvents,includingaromatic hydrocarbons, suchas'benzene and toluene, and chlorinated hydrocarbons, such as trichlorethylene, chloroform, carbon tetrachloride and perchlorethylene.

As an advantageous alternative, the flavonoids may be extracted directlyfrom the bark. Using this procedure, fl1e:hark is preferably'iirsttreated with a .defatting .solvent .to extract the fatty material andwaxes, and the resultant defatted bark dried. Treatment of the defattedbark :with the aforementioned oxygenscontaining solvents efzfectivelyextracts a mixture of fiavonoids, yielding -a fiayonoid :residue .uponevaporation of the :solvent.

,Recovery of myricetin :from the :flavonoid residue, 1 have'found, maybe effected by trituration of the fiatvonoid residue with one ,of theaforementioned oxygencQntaining :solvents. ,Due to the comparativeinsolubility of ;myricetin, triturationcf the fiavonoid residueselectiuely dissolves ;quercetin and the remaining flavanones,;leaving;an, insoluble yellow residue which is nearly pure ,myricetin.Alternatively, larger quantities of the solventcan beused to completelydissolve the flavonoid residue, and myricetinselectively crystallized bypartial evaporation of the-solution.

The myricetin may be further purified by recrystallization from asolvent. Upon recrystallization from warm acetone, the pure crystallinemyricetin recovered contained acetone of crystallization. Afterprolonged drying in vacuo, the crystalline monoacetonate of myricetin,melting at 350 C. with decomposition, may be recovered.

.Quercetin may be selectively precipitated and recovered from theremaining fiavonoids of lodgepole pine bark. Thus, when the 'fiavonoidresidue-has been triturated with a-solvent to remove quereetinandthe-remaining flavonoids, the quercetin can be selectivelyremovedfronrthe solvent. Quiercetin obtained in thismanneriscontaminated with traces of myricetin, and may be ,fractionall-yprecipitated from:a solvent. After separate .remoyal of .both myricetinand quercetin, the flavonoid residuercontains onlythe water-solublefiavanones. By the addition-of dibasic'potassiurn phosphate to an aque-,ous solution of these flavanones, the 3-hydroxyflavanones, suchasampelopsin and dihydroquercetin, are precipitated as their potassiumsalts; resuspension of the potassium salts in water, followed by theaddition of dilute hydrochloric acid causes the flavanones tocrystallize.

,In this manner, substantially all of the flavonoids con- .tained inthe-barkof lodgepole pine rnay be readily and inexpensively recovered.

In preparing myricetin according to the practice of the invention,either stainless steel or glass-lined equipment is preferred,sincetthemse of ordinary iron equipment causes appreciable discoloration,in the final product. When the iron equipment is psed, however, thisdiscoloration in the ,productmay .be removed by recrystallizing themyricetin from acetone containing a small amount of hydrochloric. acid.

'jThe' following specific examples are illustrative of the practice ofthe invention:

EXAMPLE I.ISOLATION OF FLAVONES The bark of lodgepole pine, grown in thevicinity of ,Grays Harbor, Washington,--was peeled from several logs andground in a hammer mill-t pass a ZO-mesh (Tyler standard) sieve. Theground bark was air dried, and extracted with ethyl ether in a large,borosilicate glass, Soxhlet-type extractor. The ether extract wasevaporated to dryness, placed in a paper thimble and extracted withbenzene in a Soxhletextractor to remove the fatty material and waxesfrom the preparation. After drying, the benzene-insoluble residue(containing the fiavonoids) was triturated with warm acetone andfiltered. The insoluble yellow crystalline residue consisted of nearlypure myricetin, recovered in 2.0 percent yield by weight of the drybark. The filtrate was diluted with water, and placed in anice chest,resulting in thefcnnation .of a yellow precipitate of quercetin,recovered in 0,1 .pereent by weight based on the weight of theory-bark,i

EXAMPLE II.--PURIFICATION 0F I\IYRICETIN Myricetin, obtainedfronrExample I, was crystallized from acetone, yielding yellow crystals,M. P. 350 C. with decomposition, containing acetone of crystallization.The crystals were collected, and dried in vacuo over phosphorouspentoxide at 5S .C., resulting in the crystalline monoacetonate ofmyricetin (Analysis: Caled for C H O .C HgO: C, 57.44; H, 4.29. Found:C, 57.60; H, 4.87), which was chromatographically homogeneous.

Both the acetate and hexamethoxy derivates could be prepared from thecrystalline monoacetonate of myricetin, and were identical withrespective derivatives .prepared from an authentic synthetic sample ofmyricetin.

EXAMPLE IIL-ISOLATION OF QUERCETIN Crude quercetin, obtained fromExample I, was recrystallized several times from acetone, yieldingyellow crystals melting at 310-314 C., which still contained traces ofmyricetin.

Conclusive proof of structure was established by preparing the penta-acetate derivative which was identical to an authentic sample of.penta-acetylated quercetin in melting point, mixed melting point andinfrared spectra.

EXAMPLE I'V.ISOLATION OF FLA VA 8 ONES To the filtrate remaining afterprecipitation of quercetin, .as in Example 1,, was added dibasicpotassium phos phate toprecipitate the potassium salts of theB-hydroxyflavanones. The precipitate was filtered off, resuspended in-asmall volume of water, and acidified with dilute hydrochloric acid. Uponcooling, the acidified solution yielded-colorless needles, ;M. P. 236238C., which were identified by paper chromatography as a mixture ofampelopsin '(dihydromyricetin) and dihydroquercetin in a three to oneratio.

The structure of the two compounds was further verified by conversion ofthe mixture to the corresponding fiavonols with hot 20 percent sodiumbisulfite solution, which gave a mixture of yellow flavones, M. P.308320 C. By trituration-of the fiavone mixture, both myricetin andquercetin could be recovered.

I claim:

I. The process for recovering crystalline myricetin from lodgepole pinebark which comprises extracting a mixture of flavonoids includingmyricetin'from lodgepole pine bark, dissolving the mixture of fiavonoidsin an oxygenated solvent ofthe group consisting of ethyl ether, acetone,methyl ethyl ketcne, and ethyl acetate, selectively crystallizingmyricetin from the solvent in the .presenceof the remainingdissolvedafiavonoids, and recovering crystalline rnyricetin from thesolvent.

2. The process for recovering crystalline myricetin from lodgepole pinebark which comprises extracting flavonoids and fatty material fromlodgepole pine bark, dissolvingthe fatty material of the extractin adefatting solvent of the group consisting of benzene, toluene,trichlorethylene, chloroform, carbon tetrachloride, and

.perchlorethylene while leaving the flavonoid content thereof as aresidual solid mixture of flavonoids, dissolving the mixture offlavonoids in an oxygenated solvent of the group consisting of ethylether, acetone, methyl ethylketone, and ethyl acetate, selectivelycrystallizing myricetin from the oxygenated solvent in the presence ofthe remaining dissolved flavonoids, and recovering crystalline myricetinfrom the oxygenated solvent.

3. The process for recovering crystalline myricetin from lodgepole pinebark which comprises extracting fatty material-from sub-dividedlodgepole pine bark, extractiug a; mixture of flavonoids from thedefatted bark. dissolving the mixture of flavonoids in an oxygenatedsolvent of the group consisting of ethyl ether, acetone,

methyl ethyl ketone, and ethyl acetate, selectively crystalingcrystalline myricetin from the oxygenated solvent.

4. The process for recovering crystalline myricetin from lodgepole pinebark which comprises extracting a mixture of flavonoids from sub-dividedlodgepole pine bark, recovering and drying the mixture, triturating thedry mixture of flavonoids with acetone and leaving the myricetin contentthereof undissolved while selectively dissolving the balance of theflavonoids in the triturating solvent, and separating and recoveringcrystalline myricetin from triturating solvent.

5. The process for recovering crystalline myricetin from lodgepole pinebark which comprises extracting flavonoids and fatty material fromsub-divided lodgepole pine bark, dissolving the fatty material of theextract in a defatting solvent of the group consisting of benzene,toluene, trichlorethylene, chloroform, carbon tetrachloride, andperchlorethylene while leaving the flavonoid content thereof as aresidual solid mixture of flavonoids, recovering and drying the mixture,triturating the dry mixture of flavonoids with acetone and leaving themyricetin content thereof undissolved while selectively dissolving thebalance of the flavonoids in the triturating solvent, and separating andrecovering crystalline myricetin from the triturating solvent.

6. The process for recovering crystalline myricetin from lodgepole pinebark which comprises extracting fatty material from lodgepole pine bark,extracting a mixture of flavonoids from the defatted bark, recoveringand drying the mixture, triturating the dry mixture of flavonoids withacetone and leaving the myricetin content thereof undissolved whileselectively dissolving the balance of the flavonoids in the trituratingsolvent, and separating and recovering crystalline myricetin from thetriturating solvent.

7. The process for recovering crystalline myricetin from lodgepole, pinebark which comprises extracting flavonoids and fatty material fromsub-divided lodgepole pine bark with an oxygenated solvent of the groupconsisting of ethyl ether, acetone, methyl ethyl ketone, and ethylacetate, dissolving the fatty material of the extract in a defattingsolvent of the group consisting of benzene, toluene, trichlorethylene,chloroform, carbon tetrachloride, and perchlorethylene while leaving theflavonoid content thereof as a residual solid mixture of flavonoids,dissolving the mixture of flavonoids in acetone, selectivelycrystallizing myricetin from the acetone solution in the presence of theremaining dissolved fiavonoids, and recovering crystalline myricetinfrom the acetone solution.

8. The process for recovering crystalline myricetin from lodgepole pinebark which comprises extracting fatty material from sub-dividedlodgepole pine bark with a defatting solvent of the group consisting ofbenzene, toluene, trichlorethylene, chloroform, carbon tetrachloride,and perchlorethylene, extracting a mixture of flavonoids from thedefatted bark with an oxygenated solvent of the group consisting ofethyl ether, acetone, methyl ethyl ketone, and ethyl acetate, dissolvingthe mixture of flavonoids in acetone, selectively crystallizingmyricetin from the acetone solution in the presence of the remainingdissolved flavonoids, and recovering crystalline myricetin from theacetone solution.

Eds et a1. Dec. 19, 1950 Kurth Dec. 15, 1953

1. THE PROCESS FOR RECOVERING CRYSTALLINE MYRICETIN FROM LODGEPOLE PINEBARK WHICH COMPRISES EXTRACTING A MIXTURE OF FLAVONOIDS INCLUDINGMYRICETIN FROM LODGEPOLE PINE BARK, DISSOLVING THE MIXTURE OF FLAVONOIDSIN AN OXYGENATED SOLVENT OF THE GROUP CONSISTING OF ETHYL ETHER,ACETONE, METHYL ETHYL KETONE, AND ETHYL ACETATE, SELECTIVELYCRYSTALLIZING MYRICETIN FROM THE SOLVENT IN THE PRESENCE OF THEREMAINING DISSOLVED FLAVONOIDS, AND RECOVERING CRYSTALLINE MYRICETINFROM THE SOLVENT.